Nucleic acid fragmentation and labeling has gained importance in the field of nucleic acid analysis. Fragmented and labeled nucleic acids are useful for efficient hybridization based nucleic acid analysis, particularly when hybridizing to immobilized probes, such as in multiplex detection using microarrays or bead based hybridization assays. Fragmentation of nucleic acids to generate fragments having hydroxyl groups at their 3′ termini is useful since a 3′ hydroxyl group permits extension with a template-dependent polymerase or labeling with a template-independent polymerase, which permits introduction of a label. The extension of fragmented, hybridized nucleic acid along a nucleic acid probe or template molecule by target directed nucleic acid synthesis is useful for both nucleic acid analysis and the generation of recombinant nucleic acids.
A method for controlled nucleic acid fragmentation has been previously described which is based on the incorporation of non-canonical nucleotides into a polynucleotide strand which is synthesized in vitro, followed by generation of an abasic site at the site of incorporation of a non-canonical nucleotide, permitting subsequent fragmentation of the synthesized polynucleotide and/or labeling at the abasic site. (U.S. Patent Application No. 2004/0005614; PCT Application No. WO 04/011665). The size distribution of the fragmented products may be controlled by the level of incorporation of non-canonical nucleotides. The level of incorporation, and subsequent nucleic acid fragment size, may be adjusted to provide a suitable fragment size for the particular downstream use of the fragmented products.
A process utilizing dUTP as the non-canonical nucleotide during DNA synthesis, uracil N-glycosylase (“UNG”) as the enzyme which removes the base portions of the non-canonical nucleotides to generate abasic sites, and cleavage of abasic sites with a polyamine, such as N,N′-dimethylethylenediamine, as described in U.S. Application No. 2004/0005614 and PCT Application No. WO 04/011665, results in the generation of fragmented polynucleotides with modified (“blocked”) 3′ ends that are capable of reacting with an aldehyde-reactive reagent. This process is useful for generation of fragmented and labeled nucleic acids suitable for analysis, for example, on a microarray, or fragmented nucleic acid targets suitable for immobilization.
The process of controlled fragmentation and labeling of single stranded nucleic acids is difficult to achieve using other methods, such as non-specific digestion with an enzyme such as DNase or chemical nucleotide modification, since these reactions cannot be carried out to completion without complete, or nearly complete, degradation of the single stranded nucleic acid to be fragmented and labeled. Double stranded DNA may be digested with restriction endonucleases to generate fragments of defined size distribution. Such enzymes are specific for a defined sequence content, both with respect to composition and length of a recognition site, and afford higher or lower frequency of cleavage depending on the restriction enzyme used. However, restriction endonucleases are specific for double stranded DNA and this approach does not apply to fragmentation of single stranded nucleic acid molecules.
A desirable feature for fragmented nucleic acids is the presence of a hydroxyl group at the 3′ end, which may serve as a substrate for polynucleotide synthesis with a polymerase via template-dependent or template-independent extension, or ligation with another polynucleotide. Such manipulations may facilitate introduction of a label. For example, a label may be introduced by extending from the 3′ end with terminal transferase (a template-independent polymerase) and a labeled nucleotide. The method described above, in which nucleic acid molecules are cleaved at an abasic site with a polyamine, results in nucleic acids with “blocked” 3′ ends, precluding use of the fragments as primers for polynucleotide synthesis or substrates for labeling at a 3′ hydroxyl group.
New methods for the efficient fragmentation of nucleic acids to generate fragments comprising 3′ end hydroxyl groups, and of defined size distribution, are desirable.